Red blood cells require efficient iron uptake and meticulous coordination or iron acquisition, globin protein production and heme biosynthesis. This is accomplished, at least in part, through a regulated surge in the number of cell surface transferrin receptors (TfRs) just prior to the start of hemoglobinization. Though it is widely believed that the transferring cycle is important for iron uptake by all mammalian cells, this may not be the case. Must studies of the transferring cycle have been carried out in erythroid cells and tumor cell lines. The work proposed in this application investigates the hypothesis that the primary function of the TfR is not to deliver iron ubiquitously, but rather to increase the efficiency of iron transport into erythroid cells, through a process that is tightly coupled to erythroid maturation. To this end, embryonic stem cells have been produced that carry a targeted disruption of the TfR gene. These cells have been introduced into the mouse germline, to produce mutant animals. Consistent with the hypothesis stated above, TfR-/- animals die early in gestation, apparently of severe anemia. The goal of this proposal is to characterize the role of the TfR more fully. Specific aims are (1) to determine which tissues have an absolute requirement for the TfR for normal development, (2) to understand how the TfR and transferrin cycle contribute to the regulation of erythroid differentiation and (3) to investigate the role of the TfR in regulation of body iron stores. This will be accomplished through further analysis of TfR-/- animals, through examination of the TfR mutation in the context of other mutations known to affect iron metabolism, and through in vitro differentiation and characterization of TfR-/- cells. The insights gained from this work are likely to improve our understanding of both iron deficiency and iron overload disorders.